SRY-Box transcription factor 9 triggers YAP nuclear entry via direct interaction in tumors.

The translocation of YAP from the cytoplasm to the nucleus is critical for its activation and plays a key role in tumor progression. However, the precise molecular mechanisms governing the nuclear import of YAP are not fully understood. In this study, we have uncovered a crucial role of SOX9 in the activation of YAP. SOX9 promotes the nuclear translocation of YAP by direct interaction. Importantly, we have identified that the binding between Asp-125 of SOX9 and Arg-124 of YAP is essential for SOX9-YAP interaction and subsequent nuclear entry of YAP. Additionally, we have discovered a novel asymmetrical dimethylation of YAP at Arg-124 (YAP-R124me2a) catalyzed by PRMT1. YAP-R124me2a enhances the interaction between YAP and SOX9 and is associated with poor prognosis in multiple cancers. Furthermore, we disrupted the interaction between SOX9 and YAP using a competitive peptide, S-A1, which mimics an α-helix of SOX9 containing Asp-125. S-A1 significantly inhibits YAP nuclear translocation and effectively suppresses tumor growth. This study provides the first evidence of SOX9 as a pivotal regulator driving YAP nuclear translocation and presents a potential therapeutic strategy for YAP-driven human cancers by targeting SOX9-YAP interaction.

Depletion of Tgfbr2 in hepatocytes alleviates liver fibrosis and restores hepatic function in fibrotic mice.

OBJECTIVES: Previous studies have demonstrated the pivotal role of transforming growth factor (TGF)-ß signaling in activating hepatic stellate cells during liver fibrosis. In this study we aimed to demonstrate the effects and underlying mechanism of TGF-ß signaling in hepatocytes on hepatic fibrogenesis. METHODS: Hepatocyte-specific Tgfbr2-knockout (Tgfbr2HKO ) mice were generated by AAV8-TBG-Cre injection via the tail vein of Tgfbr2f/f mice. CCl4 was injected intraperitoneally twice a week for 4 weeks to establish the fibrotic mouse model. The expression of the fibrogenesis markers was evaluated by immunohistochemistry, western blot, and real-time polymerase chain reaction (PCR). RNA-seq analysis was used to detect the transcriptional profiles of primary hepatocytes isolated from Tgfbr2HKO mice and control mice. RESULTS: The expression of TßR2 (Tgfbr2) was markedly upregulated in hepatocytes of the fibrotic liver. Tgfbr2 depletion in hepatocytes decreased the expressions of profibrogenic markers (Col1a1 and Acta2) in the CCl4 -treated fibrotic liver. RNA-seq analysis revealed that Tgfbr2 deletion in hepatocytes significantly reduced the inflammatory response and suppressed epithelial-mesenchymal transition of hepatocytes accompanied by upregulation of the metabolic pathways during liver fibrosis. Moreover, the expressions of hepatocyte nuclear factors (HNFs), including Hnf4α, Foxa1, Foxa2, and Foxa3, which are important for maintaining liver metabolism and homeostasis, were decreased in fibrotic livers and significantly increased after Tgfbr2 blockade. CONCLUSION: Blocking the TGF-ß signaling pathway in hepatocytes reduces hepatic fibrosis and improves hepatic function in fibrotic livers.

Sulodexide attenuates liver fibrosis in mice by restoration of differentiated liver sinusoidal endothelial cell.

Sulodexide is a heparinoid compound with wide-ranging pharmacological activities. However, the effect of sulodexide on liver fibrogenesis has not been reported. In this study, we aim to evaluate the therapeutic potential of sulodexide in mouse model of liver fibrosis and explore the underlying antifibrotic mechanisms. We found that sulodexide treatment significantly attenuated thioacetamide (TAA) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis in mice. Transcriptome analysis revealed that sulodexide treatment downregulated fibrosis-related genes and liver sinusoidal endothelial cells (LSECs) capillarization-associated genes in fibrotic livers. Immunohistochemistry confirmed that the increased expression of LSEC capillarization-related genes (CD34, CD31 and Laminin) in liver fibrotic tissues was reduced by sulodexide treatment. Scanning electron microscopy showed that LSECs fenestrations were preserved upon sulodexide treatment. Quantitative real-time PCR and immunofluorescence demonstrated that the expression of mesenchymal markers was downregulated by sulodexide administration, suggesting sulodexide inhibited endothelial-mesenchymal transition of LSECs during liver fibrosis. Furthermore, sulodexide administration protected primary LSECs from endothelial dysfunction in vitro. In conclusion, sulodexide attenuated liver fibrosis in mice by restoration of differentiated LSECs, indicating that sulodexide treatment may present as a potential therapy for patients with liver fibrosis.

Deletion of Sox9 in the liver leads to hepatic cystogenesis in mice by transcriptionally downregulating Sec63.

Hepatic cysts are found in heterogeneous disorders with different pathogeneses, of which simple hepatic cysts and polycystic liver diseases are two major types. The process of hepatic cytogenesis for these two diseases is caused by defects in remodelling of the ductal plate during biliary tract development, which is called ductal plate malformation. SOX9 is a transcription factor participating in the process of bile duct development, and thus, its dysregulation may play important roles in hepatic cystogenesis. SEC63 encodes an endoplasmic reticulum membrane protein that is mutated in human autosomal dominant polycystic liver disease. However, the transcriptional regulation of SEC63 is largely unknown. In the present study, a liver-specific Sox9 knockout (Sox9LKO ) mouse was generated to investigate the roles and underlying mechanism of SOX9 in hepatic cystogenesis. We found that hepatic cysts began to be observed in Sox9LKO mice at 6 months of age. The number and size of cysts increased with age in Sox9LKO mice. In addition, the characteristics of hepatic cytogenesis, including the activation of proliferation, absence of primary cilium, and disorder of polarity in biliary epithelial cells, were detected in the livers of Sox9LKO mice. RNAi silencing of SOX9 in human intrahepatic biliary epithelial cells (HIBEpic) resulted in increased proliferation and reduced formation of the primary cilium. Moreover, Sec63 was downregulated in primary biliary epithelial cells from Sox9LKO mice and SEC63 in HIBEpic transfected with siSOX9. Chromatin immunoprecipitation assays and luciferase reporter assays further demonstrated that SOX9 transcriptionally regulated the expression of SEC63 in biliary epithelial cells. Importantly, the overexpression of SEC63 in HIBEpic partially reversed the effects of SOX9 depletion on the formation of primary cilia and cell proliferation. These findings highlight the biological significance of SOX9 in hepatic cytogenesis and elucidate a novel molecular mechanism underlying hepatic cytogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Role of Bone Morphogenetic Protein 9 in Nonalcoholic Fatty Liver Disease in Mice.

Background and Aims: It's reported that bone morphogenetic protein 9 (BMP9) played an important role in lipid and glucose metabolism, but the role of BMP9 in nonalcoholic fatty liver disease (NAFLD) is unclear. Here, we evaluated the therapeutic efficacy of recombined BMP9 in NAFLD mice and investigated the potential mechanism. Methods: The effects of recombinant BMP9 on NAFLD were assessed in HFD-induced NAFLD mice. C57BL/6 mice were administrated with high-fat diet (HFD) for 12 weeks. In the last 4 weeks, mice were treated with PBS or recombined BMP9 once daily. Insulin sensitivity was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT) at the end of the 12th week. Then NAFLD related indicators were assessed by a variety of biological methods, including histology, western blotting, real-time PCR, RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses. Results: BMP9 reduced obesity, improved glucose metabolism, alleviated hepatic steatosis and decreased liver macrophages infiltration in HFD mice. RNA-seq showed that Cers6, Cidea, Fabp4 involved in lipid and glucose metabolism and Fos, Ccl2, Tlr1 involved in inflammatory response downregulated significantly after BMP9 treatment in HFD mouse liver. ATAC-seq showed that chromatin accessibility on promoters of Cers6, Fabp4, Ccl2 and Fos decreased after BMP9 treatment in HFD mouse liver. KEGG pathway analysis of dysregulated genes in RNA-seq and integration of RNA-seq and ATAC-seq showed that TNF signaling pathway and Toll-like receptor signaling pathway decreased in BMP9 treated HFD mouse liver. Conclusion: Our data revealed that BMP9 might alleviate NAFLD via improving glucose and lipid metabolism, decreasing inflammatory response and reshaping chromatin accessibility in HFD mouse liver. BMP9 downregulate genes related to lipid metabolism, glucose metabolism and inflammation expression, at least partially via decreasing promoter chromatin accessibility of Cers6, Fabp4, Fos and Tlr1. BMP9 may also reduce the expression of liver Ccl2, thereby changing the number or composition of liver macrophages, and ultimately reducing liver inflammation. The effect of BMP9 on NAFLD might be all-round, and not limit to lipid and glucose metabolism. Therefore, the underlying mechanism needs to be studied in detail further.

miR-541 potentiates the response of human hepatocellular carcinoma to sorafenib treatment by inhibiting autophagy.

OBJECTIVE: Autophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by modulating miR-541-initiated microRNA-autophagy axis. DESIGN: Gain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the malignant properties and autophagy of human HCC cells. Autophagy was quantified by western blotting of LC3, transmission electron microscopy analyses and confocal microscopy scanning of mRFP-GFP-LC3 reporter construct. Luciferase reporter assays were conducted to confirm the targets of miR-541. HCC xenograft tumours were established to analyse the role of miR-541 in sorafenib-induced lethality. RESULTS: The expression of miR-541 was downregulated in human HCC tissues and was associated with malignant clinicopathologic phenotypes, recurrence and survival of patients with HCC. miR-541 inhibited the growth, metastasis and autophagy of HCC cells both in vitro and in vivo. Prediction software and luciferase reporter assays identified autophagy-related gene 2A (ATG2A) and Ras-related protein Rab-1B (RAB1B) as the direct targets of miR-541. Consistent with the effects of the miR-541 mimic, inhibition of ATG2A or RAB1B suppressed the malignant phenotypes and autophagy of HCC cells. Furthermore, siATG2A and siRAB1B partially reversed the enhancement of the malignant properties and autophagy in HCC cells mediated by the miR-541 inhibitor. More interestingly, higher miR-541 expression predicted a better response to sorafenib treatment, and the combination of miR-541 and sorafenib further suppressed the growth of HCC cells in vivo compared with the single treatment. CONCLUSIONS: Dysregulation of miR-541-ATG2A/RAB1B axis plays a critical role in patients' responses to sorafenib treatment. Manipulation of this axis might benefit survival of patients with HCC, especially in the context of the highly pursued strategies to eliminate drug resistance.